Plants have evolved an array of cellular mechanisms to defend themselves against invading pathogens. Upon recognition of pathogens, plants activate a complex set of defense reactions to reject pathogen attack. It includes acute defense at the infection site following sometimes by induction of immunity at distal plant parts. Development of resistances at local and distal sites is associated in part with the expression of anti-microbial proteins. Those include glucanases, chitinases, and lysozymes deposited in the plants that are exhibiting increased resistance to various microorganisms. Recently, anti-microbial proteins had been introduced into crop plants in order to control phyto-pathogens. Particularly, the antifungal polypeptide can be applied directly onto susceptible plants to provide protection against fungus.
Colletotrichum gloeosporioides (Penz.) is the casual agent of anthracnose diseases, which is the most destructive disease in worldwide grown pepper. Nowadays anthracnose fungus is mainly controlled by application of substantial amounts of chemicals. To limit agro-chemicals in crop cultivation, there is an urgent need for the establishment of resistant pepper cultivars for anthracnose disease. However, classical breeding is not applicable for the development of resistance line, since there is no available genetic resource for the disease resistance. At present, all commercial pepper lines are still susceptible to anthracnose disease.
In pepper, plant responses to fungal morphogenesis are different dependent on the ripening stage of pepper fruits. Disease symptoms are developed in the unripe fruit, but not in the ripe fruits infected with anthracnose fungus. To gain genetic resources responsible for the resistance, several genes were previously isolated from the ripe fruit interacting with anthracnose. Among them, a pepper esterase gene (PepEST) encoding a member of esterase family showed an antifungal activity against anthracnose fungus and rice blast fungus. In this invention, the mode of the enzymatic action of PepEST protein was elucidated to gain an insight into the molecular mechanisms involved in the resistance reaction in pepper and fungus. PepEST protein was externally applied to induce defense reaction in the unripe fruit and to block fungal penetration into fruit epidermis.
Genetic engineering techniques are offering relief from fungal pathogens through the development of fungus control systems based on PepEST gene, which was isolated by the inventors and was disclosed in U.S. Pat. No. 6,018,038. First is the development of new fungicides using genetically engineered protein. This idea has been already disclosed in U.S. Pat. No. 6,613,323 by the inventors themselves. The second type of application is applying the protein to plants to elicit defense reactions, such as H2O2 production and PR gene expressions, and to block fungal penetration into plant cells.
The plant cuticle is a physical barrier composed of polyesters of C16 and C18 hydroxy fatty acids. Infection of C. gloeosporioides is achieved through conidium germination and appressorium formation that are necessary for subsequent cuticular penetration. The penetration of fungus into plants is associated with cutinases that hydrolyse plant cuticles. Degradation products of plant cuticles by cutinases serve as chemical signals for activating cutinase gene of fungus. Also, cutin monomers can act to induce resistance in plants against fungal infection. Spray application of C18 family of cutin monomers protected barley and rice against phytopathogenic fungi, Erysiphe gramis f. sp. Hordei and Magnaporthe grisea infection, repectively. Particularly, cutin monomers were reported as the elicitor of H2O2 and the enhancer of other H2O2 elicitors in plants. In the present work, we hypothesize that exogenous treatments of a pepper esterase would liberate cutin monomers that can elicit the generation of H2O2. Then, plant defense genes induced by H2O2 are deployed for plant protection against fungus.
Ultimately, application of PepEST proteins can provide agromonically relevant level of disease control on pepper cultivation without harmful side effects so as to contribute to more sustainable agricultural practices.